During the development of human oral biofilm communities, microbial interactions are thought to drive the spatial arrangement within bacterial communities. Such communities on enamel form supragingival dental plaque. These intimate interactions are facilitated by physical interactions called coaggregations, which are specific adherences of genetically distinct partner cells that bind to one another to form multicellular networks such as the multispecies communities of human dental plaque. In this reporting period, we used chemically pure 4,5-dihydroxy-2,3-pentanedione (DPD) that was supplied by our colleague Dr. Martin Semmelhack in the Department of Chemistry at Princeton University to show that picomolar concentrations of this diffusible signal molecule are optimal for mediating mutualistic interactions between two oral bacterial species. DPD, a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). ). AI-2 was proposed by our colleague Dr. Bonnie Bassler in the Department of Molecular Biology and the Howard Hughes Medical Institute at Princeton University to be a universal signal molecule mediating inter-species communication among bacteria. We reported that mutualistic and abundant biofilm growth in flowing saliva of two coaggregation-partner human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by chemical (synthetic AI-2 in the form of DPD) or genetic complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Significantly, in this natural two-species system, the optimal DPD concentration is 100- to 1000-fold lower than the detection limit of the currently accepted AI-2 bioassay, indicating that only a very low concentration of AI-2 is required for these organisms to conduct AI-2-signaled inter-species communication. We demonstrated for the first time that picomolar concentrations of the universal inter-species signal AI-2 mediate mutualistic interactions among members of a natural dual-species community. These studies verify that AI-2 is a bona-fide inter-species signal, and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity. The role of diffusible signaling molecules in establishing these early communities remains a topic of much interest in my laboratory.[unreadable] [unreadable] The initial colonizers of tooth surfaces are a specific subset of the oral microflora. Of these bacteria, those that colonize the clean enamel surface independently of other bacteria possess mechanisms for attachment to the acquired salivary pellicle covering the enamel and the ability to metabolize salivary components as the sole nutritional source. Alternatively, some bacterial species participate in consortia that are able to attach to enamel and establish as an initial community requiring metabolic interactions among their members. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Our hypothesis is that the initial colonization of tooth surfaces is a repeatable and selective process with certain bacterial species predominating in the nascent biofilm. In this reporting period, we used molecular methods and a retrievable enamel chip model (clinical protocol D-98-0116) to characterize the microbial diversity of early dental biofilms in three subjects. Five hundred and thirty-one 16S-rRNA-gene sequences were analyzed, and 97 distinct phylotypes were identified. The study showed that microbial community composition was statistically different among subjects and that more than two-thirds of the bacterial species in the initial dental plaque were unique to each of the three subjects in the study. A set of ten species was in common in the three subjects, and this result promoted the idea that initial colonization occurred with a subset of species particularly adapted to community development on otherwise unoccupied enamel surface. Repetitive and distinctive community composition within subjects was observed and supported our hypothesis that the initial colonization of tooth surfaces was a repeatable and selective process with certain bacterial species predominating in the nascent biofilm. [unreadable] [unreadable] Metabolic cooperation among bacteria may be important to the establishment of stable oral biofilm communities, and food webs could be set up through this cooperation. Streptococci make up 60-90% of supragingival plaque biomass in the first 24 hours of colonization; they catabolize carbohydrates to short-chain organic acids such as lactic acid and pyruvic acid. Veillonellae constitute as much as 5% of initial plaque biomass, but are unable to catabolize sugars. They rely on the fermentation of organic acids to propionic and acetic acids, carbon dioxide, and hydrogen. Thus, a rudimentary food web could exist whereby veillonellae depend upon organic acids produced by streptococci. Few data exist on proximity of veillonellae and streptococci in plaque. We hypothesized that cell-cell recognition between veillonellae and streptococci was important in supragingival plaque formation, and we set out to examine this in a human model system. The retrievable enamel chip model (clinical protocol D-98-0116) was used to investigate site-specific isolation of veillonellae and streptococci. In this reporting period, we discovered veillonellae isolates bearing adhesins that specifically recognize Type-G receptor polysaccharide (G-RPS) on streptococci. We showed that, between four and eight hours into plaque development, the dominant strains of Veillonella changed in their phenotypic characteristics (coaggregation and antibody reactivity) as well as in their genotypic characteristics (16S-RNA gene sequences and strain-level genomic fingerprint patterns). This succession was coordinated with the development of mixed-species bacterial colonies. Changes in community structure can occur very rapidly in natural biofilm development, and we suggest that this process may influence evolution within this ecosystem. The discovery of G-RPS-recognizing veillonellae is the first example of oral bacteria specifically coaggregating with G-RPS-bearing streptococci. These results suggest that these two kinds of bacteria will be found at the same sites in the oral cavity and offer an example of evolution-directed mixed-species communities. Our long-range goal is to understand the molecular mechanisms of cellular communication and their relationship to the spatiotemporal development and establishment of dental plaque and colonization of the host epithelial cells.